Project Summary Familial thrombocytopenia is a genetic disease resulting in low platelet counts and increased bleeding. A rare subset of these patients have mono-allelic mutations in either ETV6 or RUNX1 and unfortunately have a high propensity to develop acute myeloid leukemia (AML) as well as other hematologic malignancies. Patients with mutations in either of these two transcription factors display the same phenotype of small, immature megakaryocytes that subsequently give rise to fewer, less functional platelets. Although mono-allelic RUNX1 mutations have been found in approximately 40 families, the mechanism of disease is poorly understood. Mouse models have been used to study familial thrombocytopenia, but there are currently none that fully recapitulate the human disease phenotype of severely decreased platelets counts and development of leukemia. As an alternative approach, the directed differentiation of pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs), in culture can be used. The goal of this proposal is to elucidate the mechanism of familial thrombocytopenia due to mono-allelic mutations in ETV6 or RUNX1, and determine if there is a relationship between these two transcription factors, given that mono-allelic mutations in either of them lead to the same hematologic phenotype. We will use a human stem cell model to study this. In Aim 1, we will determine which stages of megakaryopoiesis and thrombopoiesis are affected due to mono-allelic mutations in ETV6 or RUNX1 through megakaryocyte colony assays, flow cytometry, proplatelet formation assays, and mouse infusion assays. Although some of these studies have already been done with patient-derived RUNX1 iPSC lines, we will confirm their findings in iPSCs from a patient with a novel mutation. The purpose of Aim 2 is to determine the mechanism through which ETV6 and RUNX1 control megakaryocyte specification and differentiation. For Aim 2a, we seek to find common direct targets of ETV6 and RUNX1, and then determine which direct targets are dysregulated when mono-allelic mutations in either gene are present. We will try rescuing the megakaryocyte and platelet defects seen in the ETV6 and RUNX1 iPSC lines by overexpressing differentially expressed target genes. Aim 2b will define the relationship between ETV6 and RUNX1. Co-immunoprecipitation and western blotting will determine if these two factors are bound to one another in the non-disease setting. We will also determine if the binding of either transcription factor is altered when a mutation is present in the other gene. The proposed experiments will clarify the role of ETV6 during megakaryopoiesis and thrombopoiesis and inform us of the relationship between ETV6 and RUNX1. The results from these studies will further our understanding of megakaryocyte biology and may have important implications for therapeutic strategies in treating familial thrombocytopenia patients, both for their thrombocytopenia and their risk of AML.